The invention provides methods and compositions for protein structure analysis, including substrate binding sites, sites of protein-protein interactions, three dimensional structure analysis, and stability, all with single amino acid resolution. In general, the subject methods involve introduction of cysteine residues, which serve as probes for physical analysis, into a protein by translational misincorporation in vivo. In many embodiments, proteins containing misincorporated cysteine residues are reacted with a crosslinking agent that covalently links misincorporated cysteine residues to a proximal amino acid in the folded protein. These methods, termed “MXLINK” methods, may be used for protein tertiary structure analysis. In other embodiments, cysteine-misincorporated proteins are used in protein footprinting methods, termed “MPAX” or “MSX” methods.
本发明提供了用于蛋白质结构分析的方法和组合物,包括底物结合位点、蛋白质-蛋白质相互作用位点、三维结构分析和稳定性,所有这些均以单
氨基酸分辨率进行。一般来说,这些方法涉及在体内通过翻译错结合将作为物理分析探针的半胱
氨酸残基引入蛋白质。在许多实施方案中,含有错结合半胱
氨酸残基的蛋白质与
交联剂反应,
交联剂将错结合半胱
氨酸残基与折叠蛋白质中的近端
氨基酸共价连接。这些被称为 "MXLINK "的方法可用于蛋白质三级结构分析。在其他实施方案中,半胱
氨酸错杂蛋白可用于蛋白质足迹分析方法,称为 "MPAX "或 "MSX "方法。